Nanopores hold great potential for genomic screening and sequencing technologies. Thus far, most studies have concentrated on the Staphylococcus aureus pore-forming protein alpha hemolysin (αHL)1 and artificial pores in solid-state (SS) membranes2. While biological pores offer an atomically precise structure3 and genetic engineering potential4, SS-pores offer durability, size and shape control5 and integratability. Each system, however, also has significant limitations: αHL is difficult to integrate because it relies on delicate lipid bilayers for mechanical support, and the fabrication of SS-pores at precise dimensions remains challenging. Here we show that these limitations may be overcome by inserting a single αHL pore into a SS-nanopore. A double-stranded DNA attached to a protein pore is threaded into a SS-nanopore by electrophoretic translocation. Protein insertion is observed in 30-40% of our attempts and translocation of single-stranded DNA demonstrates that the hybrid nanopore remains functional. The resulting hybrid structure offers a platform to create wafer-scale device arrays for genomic analysis including sequencing6.
Nanopores have been used as extremely sensitive resistive pulse sensors to detect analytes at the molecular level. There has been interest in using such a scheme to rapidly and inexpensively sequence single molecules of DNA. To establish reference current levels for adenine, cytosine, and thymine nucleotides, we measured the blockage currents following immobilization of single-stranded DNA polyadenine, polycytosine, and polythymine within a protein nanopore in chemical orientations in which either the 3' or the 5' end enters the pore. Immobilization resulted in low-noise measurements, yielding sharply defined current distributions for each base that enabled clear discrimination of the nucleotides in both orientations. In addition, we find that not only is the blockage current for each polyhomonucleotide orientation dependent, but also the changes in orientation affect the blockage currents for each base differently. This dependence can affect the ability to resolve polyadenine and polythymine; with the 5' end entering the pore, the separation between polyadenine and polythymine is double that observed for the 3' orientation. This suggests that, for better resolution, DNA should be threaded through the 5' end first in nanopore DNA sequencing experiments.
Shown below are the results from a comparison of fully methylated and unmethylated versions of the BRCA1 and MS3 genomic DNA fragments(A). Using quantitative PCR, we measured the number of DNA molecules which translocated through a 1.8nm nanopore at a given voltage(B). Unmethylated MS3 and BRCA1 translocate above 3.77V and 3.61V, respectively while the thresholds for fully methylated MS3 and BRCA1 are 2.53V and 2.69V respectively.
We have developed a mixture of enzymes and chemicals that completely lyse cyanobacteria. Since the treatment involves only readily-available chemicals and simple proteins that degrade the components of the cyanobacterial cell wall, it can easily be used in high-throughput applications requiring lysis for subsequent intracellular measurements. Our lysis technique consistently enables complete lysis of several different cyanobacterial strains, and we demonstrated that DNA, mRNA, and proteins are preserved in the lysates. Chemical lysis can be superior to existing techniques because of its convenience, reliability, and amenability to a variety of downstream applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-015-0007-y) contains supplementary material, which is available to authorized users.
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